1Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital and University of Oslo, 0310 Oslo, Norway.

Abstract

In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork. We show that this is, indeed, the case by using global ChIP on Chip analysis of SeqA in cells synchronized regarding DNA replication. To assess hemimethylation, we developed the first genome-wide method for methylation analysis in bacteria. Since loss of the SeqA protein affects growth rate only during rapid growth when cells contain multiple replication forks, a comparison of rapid and slow growth was performed. In cells with six replication forks per chromosome, the two old forks were found to bind surprisingly little SeqA protein. Cell cycle analysis showed that loss of SeqA from the old forks did not occur at initiation of the new forks, but instead occurs at a time point coinciding with the end of SeqA-dependent origin sequestration. The finding suggests simultaneous origin de-sequestration and loss of SeqA from old replication forks.

Whole genome SeqA binding in synchronized E. coli dnaC2 cells. Cells were grown exponentially in AB medium supplemented with glucose and CAA at 30°C and shifted to 39°C for 70 min. Synchronous initiation of replication was induced by a downshift to 30°C. Parts of the culture (60 ml) were removed and subjected to cross-linking at the indicated time points after downshift. ChIP-Chip was performed with a SeqA specific antibody and enrichment values are relative to values of an unsynchronized wt culture (LB) with a moving window of 60 kbp and a step size of 6 kb (). Data are plotted as a function of their chromosomal position with oriC in the middle and the distance to oriC indicated. The first pair of SeqA peaks/replication forks and the second pair after reinitiation are indicated by white and grey arrows, respectively.

SeqA binding in synchronized E. coli dnaC2 cells at 15, 16 and 17 min after initiation. The experiment and data processing was as described (). Relative values for the three time points were aligned at the oriC region based on the finding that SeqA binding there is relatively stable. Only the SeqA bound part of the chromosome is shown with oriC in the middle and the distance to oriC as indicated (for whole genome plot of 15 min time point see ). Values for the different time points are colored according to legend. The blue scale bar indicates the number of base-pairs replicated per minute in the used system according to ().

Methylation of synchronized E. coli dnaC2 cells at 15 min after initiation. Cell growth was as described (). 15 min after synchronous initiation of replication cells were collected and chromosomal DNA isolated. For details on the methylation analysis, see ‘Results’ and ‘Materials and Methods’ sections. Values shown are average enrichment factors for a moving window of 60 kbp, step size of 6 kb. Red, methylation analysis. Grey, SeqA binding data from at similar time point.

Chromosome-wide SeqA binding varies according to the replication pattern. (A)Top panel: Whole-genome plot of the SeqA binding in unsynchronized, slow-growing E. coli MG1655 cells (AB-acetate) with no overlapping replication (red) and unsynchronized, fast-growing E. coli MG1655 cells (LB-glucose) with overlapping replication (blue). The sum of SeqA ChIP signals in windows of 60 kbp are shown (step size 1 kbp; only values >1 were included). Red/blue second circle from inside: mean centred line graph with ratios of AB-acetate (red) and LB-glucose (blue). Inner circle: mean centred line graph of number of GATC sites in windows of 5 kbp (step size 1 kbp). Outer lines indicate the region replicated by the new (black) and old (grey) forks during the origin sequestration time found to be 1/3 of the cell cycle (). Outer green circle (see below for explanation): regions replicated by new and old forks at different stages of the cell cycle presented with the same colour code as in the bottom panel. Bottom panel: Schematic of the replication pattern of the cells grown in LB-glucose medium. The width of the rectangle represents one generation with the different greens indicating the different events in the life of the average cell: the period from cell birth to termination (0–14 min), from termination to initiation (14–18 min) and from initiation to cell division (18–22 min). Examples of cells (rods in shades of green) are shown above each of the three intervals with chromosomes (black lines) and origins (black dots). Horizontal lines represent the C period (black) and D period (white line; the total duration of which spans more than three generations) and should be read from top to bottom (see ‘Materials and Methods’ section for calculation details). Initiation of chromosome replication occurs in 18 min old cells leading to 16 copies of oriC (dark green). After cell division at an age of 22 min the cells contain eight oriC copies (light green). The chromosomes are then replicated with four old and eight new forks (light green cell). Old forks finish replication at 14 min cell age. (B) Examples of SeqA binding to chromosomal sites in AB-acetate (red) and LB-glucose medium (blue). Values are mean values of two replicates. Chromosomal positions of sites are marked by roman numbers in A.

New replication forks are more frequently bound by SeqA. (A) The chromosome was divided in 5 kbp windows and grouped according to their GATC content and the number of respective windows in two 1.5 Mbp regions at the origin and terminus was calculated as indicated. An example of 41 windows with 19 GATCs is marked in green. (B) The average SeqA ChIP signal was calculated for all windows in the ter or oriC region with the same number of GATC sites. Values were plotted versus each other for data derived from cells grown in LB-glucose (blue) and AB-acetate (red). The black line indicates the theoretical pattern if binding is the same in the oriC and the ter region.

SeqA binding to the GATCs cluster at different insertion sites. E. coli strains carrying cluster insertions at indicated sites were grown in LB batch culture. SeqA ChIP enrichment was quantified by qPCR with primers specific for the GATC cluster or oriC. Respective ratios of three experiments are shown with the standard deviation. The distance of the cluster insertion to oriC are indicated in Mbps.

Insertion of a GATC cluster affects SeqA binding to neighbouring sites. E. coli strains with a cluster inserted at indicated regions (arrow) of the chromosome were grown in LB-glucose batch culture. SeqA ChIP-Chip was performed in duplicate for each strain. ChIP signals are plotted for a region of 250 probes up and down stream of the insertion (∼60 kbp). Red, values for the strain with the insertion. Blue, average of the respective three other strains. Line plots below the diagrams show a rank based data analysis. Every black line marks one data point out of the 500 most enriched in the respective strain compared to the three others. Colour code as above.